The signaling cascades of the Eph receptor tyrosine kinase family and the ephrin ligands have been implicated in pathological forms of angiogenesis and tumorigenesis. While initial investigations have focused on the role of this protein-protein interaction in axonal guidance, cell attachment and motility, there is now significant evidence for the overexpression and dysregulation of this interaction in numerous cancerous tissues. The key research hypothesis of this proposal is that a limited set of structural determinants are responsible for the promiscuity and selectivity of ligand recognition exhibited by the Eph receptor family, and that these structural determinants can be exploited for small molecule modulators of this interaction. The proposed research will result in a demonstrated description and characterization of the mode of binding across this large family of receptor-ligand interactions. The outcome will be translated in a discovery approach to the development of small molecule compounds that exploit these structural determinants. Aim 1: Evaluate structural determinants of specificity versus promiscuity in Eph receptor-ligand binding by using X-ray crystallography. Aim 2: Develop strategies to stabilize the flexible Eph receptor J-K loop in the ephrin-binding cleft to facilitate receptor crystallization in complex with small molecular weight chemical compounds. Aim 3: Characterize and improve small molecular weight chemical compounds identified in high throughput screens and virtual ligand screens for inhibitors of EphB2 and EphB4 receptor ligand binding.